Computational protocol: DotU and VgrG, Core Components of Type VI Secretion Systems, Are Essential for Francisella LVS Pathogenicity

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Protocol publication

[…] The conserved domain architecture retrieval tool (CDART) was used to identify homologues of F. tularensis DotU and to investigate DUF2077 superfamily domain architectures. Based on a length criterion aimed at selecting for full length proteins, a total of 653 DotU homologues were selected for further analysis. For single-domain proteins (contain DUF2077 domain only), only proteins longer than 200 amino acids were included, and for two-domain proteins (contain DUF2077 domain and an additional OmpA or SPOR domain) only proteins longer than 350 amino acids were included. The selected homologues were aligned using MSAprobs v. 0.9.5 using 50 iterative refinement repetitions and two consistency repetitions. The conservation of the Asp70, Glu71 and Gly134 residues of the F. tularensis DotU within the dataset was investigated by visual inspection of the alignment. For efficient inference of phylogenetic relationships between the DotU homologues, the number of aligned sequences was reduced further using T-Coffee v. 8.99 , by including all Francisella DotU homologues but excluding all non-Francisella DotU homologues that exhibited more than 80% amino acid identity to any other homologue in the dataset. Thereby, a final dataset containing 283 amino acid sequences was obtained and used to determine the phylogenetic relationship among DotU proteins, which was conducted using MEGA 5.05 . Phylogenetic analysis was performed using the neighbor-joining algorithm and the Jones-Taylor-Thornton substitution model (JTT) with the pairwise deletion option . Bootstrap analysis was performed using 100 repetitions . The existence of remote homologues to F. tularensis DotU and VgrG proteins was investigated using the HHpred and Phyre2 tools, which are based on comparison of profile hidden Markov models and sequence profiles, respectively, making use of secondary structure information. […]

Pipeline specifications

Software tools CDART, MSAProbs-MPI, T-Coffee, MEGA, HHPred, Phyre
Applications Phylogenetics, Protein structure analysis, Amino acid sequence alignment
Organisms Francisella tularensis, Bacteria, Mus musculus
Diseases Tularemia